DNA Repair Proteins Research Articles

Role of senataxin in R-loop-mediated neurodegeneration.

Senataxin is an RNA:DNA helicase that plays an important role in the resolution of RNA:DNA hybrids (R-loops) formed during transcription. R-loops are involved in the regulation of biological processes such as immunoglobulin class switching, gene expression and DNA repair. Excessive accumulation of R-loops results in DNA damage and loss of genomic integrity. Senataxin is critical for maintaining optimal levels of R-loops to prevent DNA damage and acts as a genome guardian. Within the nucleus, senataxin interacts with various RNA processing factors and DNA damage response and repair proteins. Senataxin interactors include survival motor neuron and zinc finger protein 1, with whom it co-localizes in sub-nuclear bodies. Despite its ubiquitous expression, mutations in senataxin specifically affect neurons and result in distinct neurodegenerative diseases such as amyotrophic lateral sclerosis type 4 and ataxia with oculomotor apraxia type 2, which are attributed to the gain-of-function and the loss-of-function mutations in senataxin, respectively. In addition, low levels of senataxin (loss-of-function) in spinal muscular atrophy result in the accumulation of R-loops causing DNA damage and motor neuron degeneration. Senataxin may play multiple functions in diverse cellular processes; however, its emerging role in R-loop resolution and maintenance of genomic integrity is gaining attention in the field of neurodegenerative diseases. In this review, we highlight the role of senataxin in R-loop resolution and its potential as a therapeutic target to treat neurodegenerative diseases.

New ALKBH2 and ALKBH5 inhibitors for treating glioblastoma

PurposeGlioblastoma is an aggressive brain tumor with high mortality and a median survival of about 15 months. Starting from our previous published compound, MV1035 that inhibited migration and invasiveness of glioblastoma U87 cells, and also exhibited a synergic effect in combination with the alkylating agent Temozolomide, we identified new active molecules, with the aim to understand the key motifs responsible for MV1035 activity against the DNA repair protein ALKBH2 and the RNA demethylase ALKBH5. Materials and methodsWe modified the original structure of MV1035, synthesizing new molecules that have been tested through MTT assay, cell-free ALKBH2 assay and cell-free ALKBH5 assay. ResultsWe found that compound 6 is able to reduce the activity of both ALKBH2 and ALKBH5. ConclusionWe obtained important information about the key motifs responsible for the activity of these inhibitors. However, considering the complexity, heterogeneity and plasticity of GBM and GSC cells, the new identified compound will need to be validated in vivo.

DNA Repair Protein XRCC1 Stimulates Activity of DNA Polymerase λ under Conditions of Microphase Separation.

Non-membrane compartments or biomolecular condensates play an important role in the regulation of cellular processes including DNA repair. Here, an ability of XRCC1, a scaffold protein involved in DNA base excision repair (BER) and single-strand break repair, to form protein-rich microphases in the presence of DNA duplexes was discovered. We also showed that the gap-filling activity of BER-related DNA polymerase λ (Pol λ) is significantly increased by the presence of XRCC1. The stimulation of the Pol λ activity was observed only at micromolar XRCC1 concentrations, which were well above the nanomolar dissociation constant determined for the XRCC1-Pol λ complex and pointed to the presence of an auxiliary stimulatory factor in addition to protein-protein interactions. Indeed, according to dynamic light scattering measurements, the stimulation of the Pol λ activity by XRCC1 was coupled with microphase separation in a protein-DNA mixture. Fluorescence microscopy revealed colocalization of Pol λ, XRCC1, and gapped DNA within the microphases. Thus, stimulation of Pol λ activity is caused both by its interaction with XRCC1 and by specific conditions of microphase separation; this phenomenon is shown for the first time.

Ethylene glycol is metabolized to ethanol and acetate and induces expression of bacterial microcompartments in Propionibacterium freudenreichii

Ethylene glycol (EG, 1,2-ethanediol) is a two-carbon dihydroxy alcohol that can be derived from fermentation of plant-derived xylose and arabinose and which can be formed during food fermentations. Here we show that Propionibacterium freudenreichii DSM 20271 is able to convert EG in anaerobic conditions to ethanol and acetate in almost equimolar amounts. The metabolism of EG led to a moderate increase of biomass, indicating its metabolism is energetically favourable. A proteomic analysis revealed EG induced expression of the pdu-cluster, which encodes a functional bacterial microcompartment (BMC) involved in the degradation of 1,2-propanediol, with the presence of BMCs confirmed using transmission electron microscopy. Cross-examination of the proteomes of 1,2-propanediol and EG grown cells revealed PDU BMC-expressing cells have elevated levels of DNA repair proteins and cysteine biosynthesis proteins. Cells grown in 1,2-propanediol and EG also showed enhanced resistance against acid and bile salt-induced stresses compared to lactate-grown cells. Our analysis of whole genome sequences of selected genomes of BMC-encoding microorganisms able to metabolize EG with acetaldehyde as intermediate indicate a potentially broad-distributed role of the pdu operon in metabolism of EG. Based on our results we conclude EG is metabolized to acetate and ethanol with acetaldehyde as intermediate within BMCs in P. freudenreichii.

Widespread chromatin context-dependencies of DNA double-strand break repair proteins

DNA double-strand breaks are repaired by multiple pathways, including non-homologous end-joining (NHEJ) and microhomology-mediated end-joining (MMEJ). The balance of these pathways is dependent on the local chromatin context, but the underlying mechanisms are poorly understood. By combining knockout screening with a dual MMEJ:NHEJ reporter inserted in 19 different chromatin environments, we identified dozens of DNA repair proteins that modulate pathway balance dependent on the local chromatin state. Proteins that favor NHEJ mostly synergize with euchromatin, while proteins that favor MMEJ generally synergize with distinct types of heterochromatin. Examples of the former are BRCA2 and POLL, and of the latter the FANC complex and ATM. Moreover, in a diversity of human cancer types, loss of several of these proteins alters the distribution of pathway-specific mutations between heterochromatin and euchromatin. Together, these results uncover a complex network of proteins that regulate MMEJ:NHEJ balance in a chromatin context-dependent manner.

Mechanistic insight into roles of α/β-type small acid-soluble proteins, RecA, and inner membrane proteins during bacterial spore inactivation by ohmic heating.

Ohmic heating (OH) (i.e. heating by electric field) more effectively kills bacterial spores than traditional wet heating, yet its mechanism remains poorly understood. This study investigates the accelerated spore inactivation mechanism using genetically modified spores. We investigated the effects of OH and conventional heating (CH) on various genetically modified strains of Bacillus subtilis: isogenic PS533 (wild type_1), PS578 [lacking spores' α/β-type small acid-soluble proteins (SASP)], PS2318 (lacking recA, encoding a DNA repair protein), isogenic PS4461 (wild type_2), and PS4462 (having the 2Duf protein in spores, which increases spore wet heat resistance and decreases spore inner membrane fluidity). Removal of SASP brought the inactivation profiles of OH and CH closer, suggesting the interaction of these proteins with the field. However, the reemergence of a difference between CH and OH killing for SASP-deficient spores at the highest tested field strength suggested there is also interaction of the field with another spore core component. Additionally, RecA-deficient spores yielded results like those with the wild-type spores for CH, while the OH resistance of this mutant increased at the lower tested temperatures, implying that RecA or DNA are a possible additional target for the electric field. Addition of the 2Duf protein markedly increased spore resistance both to CH and OH, although some acceleration of killing was observed with OH at 50V/cm. In summary, both membrane fluidity and interaction of the spore core proteins with electric field are key factors in enhanced spore killing with electric field-heat combinations.

HGG-22. LINKS BETWEEN M6A RNA METHYLATION, R-LOOPS AND DNA DAMAGE IN PAEDIATRIC HIGH-GRADE GLIOMAS

Abstract BACKGROUND Paediatric high-grade gliomas (pHGG) are aggressive malignant tumours of the central nervous system that unfortunately carry poor prognoses. R-loops, which are DNA:RNA hybrids, are a known source of genomic instability in eukaryotic cells. The RNA strand of R-loops can be m6A methylated at double-strand break sites in order to facilitate DNA repair. Therefore, we investigated whether dysregulation of m6A methylation and R-loop homeostasis at DNA damage sites deregulates DNA repair pathways in pHGG. METHODS Immunocytochemistry was performed to assess global m6A, R-loop, γH2AX and several DNA repair protein levels in pHGG cell lines compared to human astrocytes. Colocalisation between m6A and R-loops, R-loops and γH2AX, m6A and γH2AX, R-loops and 53BP1, R-loops and PARP1, R-loops and total ATM, and R-loops and phosphorylated ATM was assessed in pHGG cell lines and human astrocytes. RESULTS Immunocytochemistry analysis revealed that levels of global m6A, R-loop, γH2AX and several DNA repair proteins were generally higher in pHGG cell lines, thereby indicating that cellular homeostasis is dysregulated. Furthermore, a high degree of colocalisation between m6A and R-loops was observed. R-loops were found to colocalise with γH2AX, and m6A was found to colocalise with γH2AX, thereby corroborating the notion that m6A methylated R-loops are likely present at DNA damage sites. Finally, R-loops were also found to colocalise with a variety of DNA repair proteins, including 53BP1, PARP1, total ATM, and phosphorylated ATM, where different Pearson’s correlation coefficient values were obtained, thereby suggesting that R-loops associate with DNA repair proteins to different extents. CONCLUSIONS The link between m6A methylation, R-loops and DNA damage in relation to the pathogenesis of pHGG is currently being investigated. Experiments are also being performed where we are investigating m6A readers to see whether their aberrant expression contributes to disease pathogenesis via deregulating DNA repair pathways.

What makes Candida auris pan-drug resistant? Integrative insights from genomic, transcriptomic, and phenomic analysis of clinical strains resistant to all four major classes of antifungal drugs.

The global epidemic of drug-resistant Candida auris continues unabated. We do not know what caused the unprecedented appearance of pan-drug resistant (PDR) Candida auris strains in a hospitalized patient in New York; the initial report highlighted both known and unique mutations in the prominent gene targets of azoles, amphotericin B, echinocandins, and flucytosine antifungal drugs. However, the factors that allow C. auris to acquire multi-drug resistance and pan-drug resistance are not known. Therefore, we conducted a comprehensive genomic, transcriptomic, and phenomic analysis to better understand PDR C. auris . Among 1,570 genetic variants in drug-resistant C. auris , 299 were unique to PDR strains. The whole genome sequencing results suggested perturbations in genes associated with nucleotide biosynthesis, mRNA processing, and nuclear export of mRNA. Whole transcriptome sequencing of PDR C. auris revealed two genes to be significantly differentially expressed - a DNA repair protein and DNA replication-dependent chromatin assembly factor 1. Of 59 novel transcripts, 12 candidate transcripts had no known homology among expressed transcripts found in other organisms. We observed no fitness defects among multi-drug resistant (MDR) and PDR C. auris strains grown in nutrient-deficient or - enriched media at different temperatures. Phenotypic profiling revealed wider adaptability to nitrogenous nutrients with an uptick in the utilization of substrates critical in upper glycolysis and tricarboxylic acid cycle. Structural modelling of 33-amino acid deletion in the gene for uracil phosphoribosyl transferase suggested an alternate route in C. auris to generate uracil monophosphate that does not accommodate 5-fluorouracil as a substrate. Overall, we find evidence of metabolic adaptations in MDR and PDR C. auris in response to antifungal drug lethality without deleterious fitness costs.

MODL-03. THERAPEUTIC EFFICACY OF IMIPIRIDONES IN LYNCH SYNDROME-ASSOCIATED GLIOMAS

Abstract INTRODUCTION Lynch Syndrome, a hereditary cancer syndrome linked to DNA mismatch repair gene mutations (MMRD), predisposes individuals to various cancers, including high grade gliomas (Lynch-HGG). Effective treatments for Lynch-HGGs are scarce, and suitable pre-clinical models are needed to identify novel therapeutic combinations. METHODS In this study, we generated patient-derived cell line models of Lynch-HGGs (n=3), characterized them through Western blot analysis for MMRD gene markers, and conducted genome and transcriptomic analyses. Murine models were then generated via intracranial injection of cultured cells expressing mCherry-luciferase reporter genes. Drug screening based on transcriptomic analysis was performed to identify potential therapeutic agents. RESULTS Our findings showed loss of MMRD protein consistent with the primary mutation, and a constant loss of P53, a tumor suppressor protein. One cell line, 7316-3058, exhibited an absence of NF1 expression, another critical tumor suppressor protein. Transcriptomic analysis unveiled upregulation in cell cycle processes and SUMOylation of DNA damage response and repair proteins. In vitro treatment with imipiridone compounds (ONC201 and ONC206) demonstrated varying degrees of drug response, with 7316-3058 displaying high sensitivity to the targeted inhibitor. Unfortunately, this therapeutic efficacy did not translate to the in vivo murine model, suggesting underlying mechanisms that impede drug efficiency. CONCLUSION In summary, we developed effective pre-clinical models of Lynch-HGG that can be used to screen different drug combinations targeted to treat these tumors. Furthermore, despite promising in-vitro data, we demonstrated the limited efficacy of imipiridones in treating Lynch-HGG in vivo, emphasizing the challenges in translating in vitro findings to in vivo contexts. This study contributes valuable insights to the development of effective therapeutic strategies for Lynch Syndrome-associated gliomas.

PCNA molecular recognition of different PIP motifs: Role of Tyr211 phosphorylation

The coordination of enzymes and regulatory proteins for eukaryotic DNA replication and repair is largely achieved by Proliferating Cell Nuclear Antigen (PCNA), a toroidal homotrimeric protein that embraces the DNA duplex. Many proteins bind PCNA through a conserved sequence known as the PCNA interacting protein motif (PIP). PCNA is further regulated by different post-translational modifications. Phosphorylation at residue Y211 facilitates unlocking stalled replication forks to bypass DNA damage repair processes but increasing nucleotide misincorporation. We explore here how phosphorylation at Y211 affects PCNA recognition of the canonical PIP sequences of the regulatory proteins p21 and p15, which bind with nM and μM affinity, respectively. For that purpose, we have prepared PCNA with p-carboxymethyl-L-phenylalanine (pCMF, a mimetic of phosphorylated tyrosine) at position 211. We have also characterized PCNA binding to the non-canonical PIP sequence of the catalytic subunit of DNA polymerase δ (p125), and to the canonical PIP sequence of the enzyme ubiquitin specific peptidase 29 (USP29) which deubiquitinates PCNA. Our results show that Tyr211 phosphorylation has little effect on the molecular recognition of p21 and p15, and that the PIP sequences of p125 and USP29 bind to the same site on PCNA as other PIP sequences, but with very low affinity.

Interplay between altered metabolism and DNA damage and repair in ovarian cancer.

Ovarian cancer is the most lethal gynecological malignancy and is often associated with both DNA repair deficiency and extensive metabolic reprogramming. While still emerging, the interplay between these pathways can affect ovarian cancer phenotypes, including therapeutic resistance to the DNA damaging agents that are standard-of-care for this tumor type. In this review, we will discuss what is currently known about cellular metabolic rewiring in ovarian cancer that may impact DNA damage and repair in addition to highlighting how specific DNA repair proteins also promote metabolic changes. We will also discuss relevant data from other cancers that could be used to inform ovarian cancer therapeutic strategies. Changes in the choice of DNA repair mechanism adopted by ovarian cancer are a major factor in promoting therapeutic resistance. Therefore, the impact of metabolic reprogramming on DNA repair mechanisms in ovarian cancer has major clinical implications for targeted combination therapies for the treatment of this devastating disease.

ALKBH5-mediated m6A demethylation of Runx2 mRNA promotes extracellular matrix degradation and intervertebral disc degeneration

BackgroundN6-methyladenosine (m6A) methylation is a prevalent RNA modification implicated in various diseases. However, its role in intervertebral disc degeneration (IDD), a common cause of low back pain, remains unclear.ResultsIn this investigation, we explored the involvement of m6A demethylation in the pathogenesis of IDD. Our findings revealed that ALKBH5 (alkylated DNA repair protein AlkB homolog 5), an m6A demethylase, exhibited upregulation in degenerative discs upon mild inflammatory stimulation. ALKBH5 facilitated m6A demethylation within the three prime untranslated region (3′-UTR) of Runx2 mRNA, consequently enhancing its mRNA stability in a YTHDF1 (YTH N6-methyladenosine RNA binding protein F1)-dependent manner. The subsequent elevation in Runx2 expression instigated the upregulation of ADAMTSs and MMPs, pivotal proteases implicated in extracellular matrix (ECM) degradation and IDD progression. In murine models, subcutaneous administration of recombinant Runx2 protein proximal to the lumbar disc in mice elicited complete degradation of intervertebral discs (IVDs). Injection of recombinant MMP1a and ADAMTS10 proteins individually induced mild to moderate degeneration of the IVDs, while co-administration of MMP1a and ADAMTS10 resulted in moderate to severe degeneration. Notably, concurrent injection of the Runx2 inhibitor CADD522 with recombinant Runx2 protein did not result in IVD degeneration in mice. Furthermore, genetic knockout of ALKBH5 and overexpression of YTHDF1 in mice, along with lipopolysaccharide (LPS) treatment to induce inflammation, did not alter the expression of Runx2, MMPs, and ADAMTSs, and no degeneration of the IVDs was observed.ConclusionOur study elucidates the role of ALKBH5-mediated m6A demethylation of Runx2 mRNA in activating MMPs and ADAMTSs, thereby facilitating ECM degradation and promoting the occurrence of IDD. Our findings suggest that targeting the ALKBH5/Runx2/MMPs/ADAMTSs axis may represent a promising therapeutic strategy for preventing IDD.

KBM-mediated interactions with KU80 promote cellular resistance to DNA replication stress in CHO cells

The KU heterodimer (KU70/80) is rapidly recruited to DNA double-strand breaks (DSBs) to regulate their processing and repair. Previous work has revealed that the amino-terminal von Willebrand-like (vWA-like) domain in KU80 harbours a conserved hydrophobic pocket that interacts with a short peptide motif known as the Ku-binding motif (KBM). The KBM is present in a variety of DNA repair proteins such as APLF, CYREN, and Werner protein (WRN). Here, to investigate the importance of KBM-mediated protein-protein interactions for KU80 function, we employed KU80-deficient Chinese Hamster Ovary (Xrs-6) cells transfected with RFP-tagged wild-type human KU80 or KU80 harbouring a mutant vWA-like domain (KU80L68R). Surprisingly, while mutant RFP-KU80L68R largely or entirely restored NHEJ efficiency and radiation resistance in KU80-deficient Xrs-6 cells, it failed to restore cellular resistance to DNA replication stress induced by camptothecin (CPT) or hydroxyurea (HU). Moreover, KU80-deficient Xrs-6 cells expressing RFP-KU80L68R accumulated pan-nuclear γH2AX in an S/G2-phase-dependent manner following treatment with CPT or HU, suggesting that the binding of KU80 to one or more KBM-containing proteins is required for the processing and/or repair of DNA ends that arise during DNA replication stress. Consistent with this idea, depletion of WRN helicase/exonuclease recapitulated the CPT-induced γH2AX phenotype, and did so epistatically with mutation of the KU80 vWA-like domain. These data identify a role for the KBM-binding by KU80 in the response and resistance of CHO cells to arrested and/or collapsed DNA replication forks, and implicate the KBM-mediated interaction of KU80 with WRN as a critical effector of this role.

Functional Analysis of Stress Resistance of Bacillus cereus SCL10 Strain Based on Whole-Genome Sequencing.

A Gram-positive, rod-shaped, aerobic, motile, and spore-forming bacterium, designated SCL10, was isolated from Acaudina molpadioides exposure to Co-60 radiation. In this study, whole-genome sequencing was performed to identify the strain as Bacillus cereus and functional characterization, with a focus on stress resistance. The genome of the B. cereus SCL10 strain was sequenced and assembled, revealing a size of 4,979,182 bp and 5167 coding genes. The genes involved in biological functions were annotated by using the GO, COG, KEGG, NR, and Swiss-Prot databases. The results showed that genes related to alkyl hydroperoxide reductase (ahpC, ahpF), DNA-binding proteins from starved cells (dps), spore and biofilm formation (spoVG, spo0A, gerP), cold shock-like protein (cspC, cspE), ATP-dependent chaperone (clpB), and photolyase, small, acid-soluble spore protein (SASP) and DNA repair protein (recA, radD) could explain the stress resistance. These findings suggest that antioxidant activity, sporulation, biofilm formation, and DNA protection may be considered as the main resistance mechanisms under exposure to radiation in the B. cereus SCL10 strain.

Pre-pubertal obesity compromises ovarian oxidative stress, DNA repair and chemical biotransformation

Obesity in adult females impairs fertility by altering oxidative stress, DNA repair and chemical biotransformation. Whether prepubertal obesity results in similar ovarian impacts is under-explored. The objective of this study was to induce obesity in prepubertal female mice and assess puberty onset, follicle number, and abundance of oxidative stress, DNA repair and chemical biotransformation proteins basally and in response to 7,12-dimethylbenz(a)anthracene (DMBA) exposure. DMBA is a polycyclic aromatic hydrocarbon that has been shown to be ovotoxic. Lactating dams (C57BL6J) were fed either a normal rodent containing 3.5% kCal from fat (lean), or a high fat diet comprised of 60% kCal from fat, and 9% kCal from sucrose. The offspring were weaned onto the diet of their dam and exposed at postnatal day 35 to either corn oil or DMBA (1 mg/kg) for 7 d via intraperitoneal injection. Mice on the HFD had reduced (P < 0.05) age at puberty onset as measured by vaginal opening but DMBA did not impact puberty onset. Heart, spleen, kidney, uterus and ovary weight were increased (P < 0.05) by obesity and liver weight was increased (P < 0.05) by DMBA exposure in obese mice. Follicle number was largely unaffected by obesity or DMBA exposure, with the exception of primary follicle number, which were higher (P < 0.05) in lean DMBA exposed and obese control relative to lean control mice. There were also greater numbers (P < 0.05) of corpora lutea in obese relative to lean mice. In lean mice, DMBA exposure reduced (P < 0.05) the level of CYP2E1, EPHX1, GSTP1, BRCA1, and CAT but this DMBA-induced reduction was absent in obese mice. Basally, obesity reduced (P < 0.05) the abundance of CYP2E1, EPHX1, GSTP1, BRCA1, SOD1 and CAT. There was greater (P < 0.05) fibrotic staining in obese DMBA-exposed ovaries and PPP2CA was decreased (P < 0.05) in growing follicles by both obesity and DMBA exposure. Thus, prepubertal obesity alters the capacity of the ovary to respond to DNA damage, ovotoxicant exposure and oxidative stress.

Inhibition of AR and DNA-PK for radio-sensitization of AR-driven cancers.

e12593 Background: Breast cancer (BC) is the most diagnosed cancer globally with over 2.2 million new cases and 680,000 deaths annually. Radiotherapy (RT) is essential for curative BC treatment; however, 20% of BC cases are radio-resistant, with the underlying mechanisms remaining poorly understood. Previous studies in prostate cancer (PCa) have shown that androgen receptor (AR) promotes radio-resistance (RT-resistance) through transcriptional regulation and increased DNA repair capacity, including through upregulation of DNA repair proteins DNA-PK and PARP1. Although AR is expressed in 60% of BCs, the implications of AR in BC RT-resistance are not yet fully characterized. We hypothesized that AR may drive RT-resistance in BC through a similar AR-DNA repair loop. Methods: To characterize the repertoire of cofactors that cooperate with AR on the TF complex, we exposed a BC cell line to RT and performed rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME) proteomics. We identified several candidate proteins that interact with AR to further investigate as targets for RT-sensitization. Survival analyses in BC and PCa cell lines were performed to identify RT-sensitizing agents. Molecular mechanisms of RT-sensitization were assessed using ATAC-seq and RNA-seq analyses. Results: Using RIME, we identified several DNA repair proteins including DNA-PK, PARP-1, and XRCC5 as differentially recruited to chromatin-bound AR. This suggests a cooperative role and provides potential druggable targets to improve RT-sensitivity. Based on these results, we also performed a survival analysis in AR-driven BC and PCa cell lines and showed that inhibition of either AR or DNA-PK led to RT-sensitization, and dual inhibition provided an additive effect. We sought to characterize the molecular mechanisms driving the RT-sensitizing effects of AR and DNA-PK inhibition and performed ATAC-seq and RNA-seq on treated and untreated cells. Combining enzalutamide with a DNA-PK inhibitor altered expression of several pathways implicated in RT-sensitization, although these pathways were largely distinct in BC and PCa. Further, we identified distinct genes that may be responsible for this phenotype in AR-driven BC. Additionally, we found a larger effect in chromatin rearrangement after treatment in PCa than BC. Conclusions: Identifying mechanisms of RT-resistance in hormone-driven cancers is essential to develop therapeutic strategies and improve patient outcomes. This study provides key evidence for AR-driven RT-resistance in BC and PCa, and identifies DNA-PK inhibition as a potential drug target. Distinct mechanisms appear to be driving this phenotype across BC and PCa, in our models. Understanding the cooperation of DNA repair and hormone signaling pathways will help support the use of DNA-damaging agents for hormone-driven cancers, and potentially inform rational design of novel drug-RT combinations.

Recurrent Duplication and Diversification of a Vital DNA Repair Gene Family Across Drosophila.

Maintaining genome integrity is vital for organismal survival and reproduction. Essential, broadly conserved DNA repair pathways actively preserve genome integrity. However, many DNA repair proteins evolve adaptively. Ecological forces like UV exposure are classically cited drivers of DNA repair evolution. Intrinsic forces like repetitive DNA, which also imperil genome integrity, have received less attention. We recently reported that a Drosophila melanogaster-specific DNA satellite array triggered species-specific, adaptive evolution of a DNA repair protein called Spartan/MH. The Spartan family of proteases cleave hazardous, covalent crosslinks that form between DNA and proteins ("DNA-protein crosslink repair"). Appreciating that DNA satellites are both ubiquitous and universally fast-evolving, we hypothesized that satellite DNA turnover spurs adaptive evolution of DNA-protein crosslink repair beyond a single gene and beyond the D. melanogaster lineage. This hypothesis predicts pervasive Spartan gene family diversification across Drosophila species. To study the evolutionary history of the Drosophila Spartan gene family, we conducted population genetic, molecular evolution, phylogenomic, and tissue-specific expression analyses. We uncovered widespread signals of positive selection across multiple Spartan family genes and across multiple evolutionary timescales. We also detected recurrent Spartan family gene duplication, divergence, and gene loss. Finally, we found that ovary-enriched parent genes consistently birthed functionally diverged, testis-enriched daughter genes. To account for Spartan family diversification, we introduce a novel mechanistic model of antagonistic coevolution that links DNA satellite evolution and adaptive regulation of Spartan protease activity. This framework promises to accelerate our understanding of how DNA repeats drive recurrent evolutionary innovation to preserve genome integrity.

Clinical phosphoproteomics reveals aberrant signalling pathways and kinases in treatment-resistant cervical cancer.

e17529 Background: Cisplatin-based concurrent chemoradiation therapy (CCRT) is the standard for locally advanced cervical cancer. While cisplatin stands out as a highly effective chemotherapeutic option for cervical cancer, resistance to the drug often triggers relapses. Moreover, previous studies have highlighted the detrimental impact of cisplatin's toxicity, which extends beyond treatment efficacy to influence the overall prognosis of patients. Combination treatment with drugs targeting different molecular pathways increases the effectiveness of the treatment by disrupting multiple critical processes that cancer cells rely on for survival. Herein, we analyzed the phosphoproteomic profile of cervical cancer patients resistant to concurrent chemoradiotherapy to identify the crucial kinases responsible for therapeutic resistance. Methods: We used mass spectrometry for phosphoproteomic analysis to study signaling pathways and dysregulated kinases in CCRT resistance. Biopsy samples collected pre-therapy were processed by protein extraction, digestion, and labeling with 10-plex TMT. Peptides were fractionated by basic pH liquid chromatography, enriched using IMAC, and analyzed on an Orbitrap-Fusion-Tribrid mass spectrometer. Data analysis was performed using Proteome Discoverer and various bioinformatics and statistical methods. Results: The phosphoproteomic analysis revealed distinct phosphorylation signatures between the responders and non-responders. In concordance with the phosphoproteomic data the expression of phospho-SMC1A and CSNK2A1 is preserved in non-responder cohort of cervical cancer patients in concordance with the phosphoproteomic data. Further, the pathway analysis showed elevated presence of the DNA repair pathway in the treatment-resistant cohort. Furthermore, dysregulation in the phosphorylation of key DNA repair proteins, such as SMC1A, HMGN1, MGMT, DDB2, and MSH6, suggests their involvement in treatment. The kinase prediction analysis suggested the activation of kinases such as CSNK2A1, PRKDC, PLK1, NEK2 and ATM in non-responders. Subsequently, cell line based functional experiments indicated the potential of CSNK2A1 inhibitor silmitasertib in sensitizing the cells to cisplatin treatment. Conclusions: Phosphoproteomicanalysis of the cervical cancer revealed distinct phosphorylation signature and kinases involved in treatment resistance. Our findings indicated that dysregulated phosphorylation and increased expression of the kinase CSNK2A1 play a pivotal role in the treatment response of cervical cancer patients. Moreover, CSNK2A1 can sensitizes the cells to cisplatin uncovering the potential of combination treatment strategies. Our study uncovers the potential of clinical phosphoproteomics to design tailored treatment strategies in patients resistant to standard treatments.

Evolved histone tail regulates 53BP1 recruitment at damaged chromatin

The master DNA damage repair histone protein, H2AX, is essential for orchestrating the recruitment of downstream mediator and effector proteins at damaged chromatin. The phosphorylation of H2AX at S139, γH2AX, is well-studied for its DNA repair function. However, the extended C-terminal tail is not characterized. Here, we define the minimal motif on H2AX for the canonical function in activating the MDC1-RNF8-RNF168 phosphorylation-ubiquitination pathway that is important for recruiting repair proteins, such as 53BP1 and BRCA1. Interestingly, H2AX recruits 53BP1 independently from the MDC1-RNF8-RNF168 pathway through its evolved C-terminal linker region with S139 phosphorylation. Mechanistically, 53BP1 recruitment to damaged chromatin is mediated by the interaction between the H2AX C-terminal tail and the 53BP1 Oligomerization-Tudor domains. Moreover, γH2AX-linker mediated 53BP1 recruitment leads to camptothecin resistance in H2AX knockout cells. Overall, our study uncovers an evolved mechanism within the H2AX C-terminal tail for regulating DNA repair proteins at damaged chromatin.

Mechanism of Action of KL-50, a Candidate Imidazotetrazine for the Treatment of Drug-Resistant Brain Cancers.

Aberrant DNA repair is a hallmark of cancer, and many tumors display reduced DNA repair capacities that sensitize them to genotoxins. Here, we demonstrate that the differential DNA repair capacities of healthy and transformed tissue may be exploited to obtain highly selective chemotherapies. We show that the novel N3-(2-fluoroethyl)imidazotetrazine "KL-50" is a selective toxin toward tumors that lack the DNA repair protein O6-methylguanine-DNA-methyltransferase (MGMT), which reverses the formation of O6-alkylguanine lesions. We establish that KL-50 generates DNA interstrand cross-links (ICLs) by a multistep process comprising DNA alkylation to generate an O6-(2-fluoroethyl)guanine (O6FEtG) lesion, slow unimolecular displacement of fluoride to form an N1,O6-ethanoguanine (N1,O6EtG) intermediate, and ring-opening by the adjacent cytidine. The slow rate of N1,O6EtG formation allows healthy cells expressing MGMT to reverse the initial O6FEtG lesion before it evolves to N1,O6EtG, thereby suppressing the formation of toxic DNA-MGMT cross-links and reducing the amount of DNA ICLs generated in healthy cells. In contrast, O6-(2-chloroethyl)guanine lesions produced by agents such as lomustine and the N3-(2-chloroethyl)imidazotetrazine mitozolomide rapidly evolve to N1,O6EtG, resulting in the formation of DNA-MGMT cross-links and DNA ICLs in healthy tissue. These studies suggest that careful consideration of the rates of chemical DNA modification and biochemical DNA repair may lead to the identification of other tumor-specific genotoxic agents.